Treatment of gelatin with nucleic acid-degrading enzymes



Feb. 17, 1970 G. RUSSELL TREATMENT 0F GELATIN WITH NUCLEIC ACID-DEGRADING ENZYMES l Filed March 7, 1967 United States Patent O U.S. Cl. 195-4 7 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a process for preparing inert gelatin in which a natural gelatin is treated to degrade the restrainers, and the degraded products, normally the degraded nucleic acids, are removed to produce inert gelatin suitable for photographic use.

This invention relates to improvements in or relating to photographic gelatin, and is especially concerned with the purification of gelatin to give inert gelatin.

There are two main sources for the supply of gelatin. These are from bones such as of cattle and from hide such as of cattle, although it can also be obtained from pig skin. Natural gelatin is extracted from these sources and it is found that substances known as restrainers are extracted together with the gelatin and are therefore present as impurities in the natural gelatin. The quantities of these restrainers can vary widely from one natural gelatin to another and also depend very critically upon the extraction process.

When such natural gelatin is used in the preparation of silver salide photographic emulsions these restrainers exert a powerful slowing down effect upon the rate of sensitivity increase and upon the fog formation during the chemical ripening or digestion of the silver halide crystals. In addition they can influence to a lesser extent the rate of growth of the silver halide crystals during the physical ripening.

Because of the wide variations in the quantities of the restrainers in different batches and in gelatin from different sources it is very diicult to prepare silver halide photographic emulsions by xed procedures. It is therefore very important to eliminate these restrainers as far as possible from natural gelatin and so prepare a socalled inert gelatin.

It has been discovered that, by very careful processing of the bones, one can prepare a gelatin which is inert or substantially free from restrainers, i.e. the chemical sensitization proceeds at a standard rate for a given quantity of chemical sensitizer in each batch of inert gelatin. The preparation of such gelatin, however, is only possible from very carefully controlled raw materials and processes.

It has been shown that these restrainers are principally nucleic acids or fragments from such acids and it has recently been discovered that the most active restrainers are the nucleic acids of fairly high molecular weight (Journal of Photographic Science, I anuary/ February 1966. p. 9; G. Russell and D. Oliff).

Adsorbents have been used to remove impurities from gelatin, but the simple method of treating the gelatin by passing a solution of it through a column of the adsorbent, such as animal charcoal, is very ineicient in removing these nucleic acid restrainers. Even the use of uneconomically large quantities of the adsorbent will only remove at the most about half of the quantity of nucleic acids from a normal gelatin.

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According to the invention it has surprisingly been found that if the nucleic acids are degraded into smaller molecules then substantially all the restrainers can be iemoved from natural gelatin by the straightforward and simple treatment with an adsorber such as animal charcoal. One means for degrading the nucleic acids is by treatment with enzymes, a suitable example of which is deoxyribonuclease. By operating in this manner we have found that it is possible to remove the nucleic acids substantially completely so that the usual methods for determining the nucleic acids in gelatin can no longer detect their presence. The natural gelatin treated in accordance with the invention can be gelatin derived from any source. Thus, even the gelatin prepared from hide, which is normally a material containing a large proportion of restrainers, can be treated in accordance with the invention to give an inert gelatin.

We have found that the enzyme degradation of the nucleic acids works even in the presence of common gelatin impurities, such as sulphur dioxide. Preferably, however, a catalyzing metallic ion, such as the magnesium ion, is present during the treatment with the enzyme.

The invention will now be illustrated with reference t0 the following example.

EXAMPLE A highly-restrained hide gelatin (10 g.) was dissolved in Water ml.) brought to a temperature of 37 C. Deoxyribonuclease I (0.32 mg.) (ex pancreas) and magnesium sulphate (0.5 g.) were then added and the mixture was digested at 37 C. for four hours.

This solution was then passed through a column (2 x 35 cm.) containing granular animal charcoal (45 g.) and jacketed so as to be kept at a temperature of 37 C. The flow rate was about 50 ml. per hour. After each hour, a sample was taken and an ultra violet spectrum taken to ensure that the column had not been overloaded.

The solution passed through the column was treated with a little sodium acetate, and the gelatin was precipitated by the addition of ethanol, the precipitated gelatin being separated and dried in cold air.

Analysis of the gelatin showed that adenine, guanine and cytosine (some of the hydrolysis products of the nucleic acids) had been reduced to very low or zero levels. The method used to determine these bases was that described by G. Russell and D. L. Oliff in Journal of Photographic Science, vol. 14 March/April 1966.

The gelatin prepared in this way was then used as the digestion gelatin for an octahedral iodobromide emulsion of mean grain size about 1.3 and containing 5.0 mol percent of iodide. In order to check the properties of the gelatin prepared 4in accordance with the invention its sensitivity or photographic speed was checked against the time of chemical ripening or digestion. Also the density of fogging of the photographic lm was checked against the digestion time. On the accompanying drawing, which is a graph of the speed and fog density against the digestion time, the curves 1 and 1A show respectively the relationship of the speed and the fog density to the digestion time for a photographic emulsion using a gelatin prepared in accordance with the invention.

On the same graph are shown the resutls which were obtained when the untreated highly-restrained hide gelatin was used in the emulsion and when an inert bone gelatin purified conventionally were tested in the same manner. The lines 2 and 2A relate to the untreated hide gelatin and the lines 3 and 3A relate to the inert bone gelatin.

It will be seen from the graph how closely similar a gelatin prepared in accordance with the invention (lines 1 and 1A) is to a conventionally purified inert gelatin (lines 3 and 3A) since it gives a very similar photographic speed for each particular digestion rate. The gelatin can be prepared in accordance with the invention, however, far more simply and cheaply than conventional inert gelatins. It can also be seen from the graph by comparing lines 1 and 2 how the removal of the restrainers provides an inert gelatin which is suitable for use in silver halide photographic emulsions since considerably less digestion time is necessary to achieve the same sensitivity.

Various other embodiments of the present invention will be apparent to those skilled in the art without depart* ing from the scope thereof.

The embodiments of the invention in which an exclusion property or privilege is claimed are dened as follows:

1. A process for the preparation of an inert gelatin for photographic use in which a natural gelatin containing nucleic acid restrainers is treated with a. nucleic acid degrading enzyme so as to degrade a substantial proportion of said restrainers which are then removed to provide inert gelatin.

2. A process as claimed in claim 1 in which the restrainers are degraded with a nucleic acid degrading enzyme which does not react with the gelatin.

3. A process as claimed in claim 2 in which the enzyme 'is deoxyribonuclease.

4. A process as claimed in claim 2 in which a eatalyzing metal ion is present during the treatment with the enzyme.

5. A process as claimed in claim 4 in which the catalyzing metal ion is the magnesium ion.

6. A process as claimed in claim 1 in which said restrainers, after degradation thereof by said nucleic acid degrading enzyme, are removed from the gelatin Lby treatment with an absorber therefor.

7. A process as claimed in claim 6 in which the adsorber is animal charcoal.

Russell et al.: Journal of Photographic Science, vol. 14, No. l, January/February 1966, pp. 9-22.

LIONEL M. SHAPIRO, Primary Examiner U.S. Cl. XR. 96-94 

